Genomic DNA Miniprep

Genomic DNA Miniprep

 

 

• grow 5 ml cells over night at 30 °C
• spin, wash once with 1 ml H₂ O
• resuspend in 500 µl lysis buffer
• add acid washed glass beads to 1.25 ml
• vortex 2 min
• recover liquid phase with blue tip or
  punch whole into the bottom of the tube and centrifuge into another tube
• add 275 µl 7 M Ammonium acetate pH 7.0
• incubate 5 min at 65 °C , then 5 min on ice
• add 500 µl Chloroform , vortex, spin 2 min in microfuge
• take supernatant and precipitate with 1 ml Isopropanol
• incubate 5 min at RT , spin 5 min
• wash pellet with 70 % EtOH , dry and dissolve in 50 µl H₂ O
  
  
• for Southern : digest 5 µl DNA
• for PCR : use 0.5 to 1 µl DNA

 

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Buffers:

 lysis buffer:
100 mM Tris pH 8.0; 50 mM EDTA; 1 % SDS.
[for 50 ml : 5 ml 1 M Tris; 5 ml 0.5 M EDTA; 5 ml 10 % SDS]